Nerve growth factor (NGF) is required for maturation of sympathetic and sensory neurons of the peripheral nervous system (PNS). Absence of NGF signaling results in programmed cell death or apoptosis, a fate met by approximately half of PNS neuron population during development as they compete for a limited supply of NGF. Apoptosis is also prevalent in various neurodegenerative diseases, including Alzheimer's and Parkinson's disease and the pathways and factors involved are nearly identical between depelopmental and pathological apoptosis. My long term goal is to use the NGF-withdrawal model, which includes neuronal PC12 cells and sympathetic neurons, to study a novel pro-apoptotic gene, Trib3. I have already determined that Trib3 leads to neuronal PC12 cell death. Neuronal PC12 cells are NGF dependent and behave like sympathetic neurons. My first aim is to determine whether Trib3 can kill sympathetic neurons, which will be explored by Trib3 overexpression and RNAi knockdown. Second goal is to determine whether Trib3 works in parallel with Bim, another proapoptotic gene that has been studied very well. This will be explored by co-expressing Trib3 and Bim and also by Trib3/Bim double knockdown RNAi experiments. My third aim is to explore the mechanism by which Trib3 kills neuronal PC12 cells and sympathetic neurons. My preliminary data indicate that Akt, an important cell survival molcule, may be a target of Trib3. I will explore this by Trib3 overexpression and RNAi knockdown and use western blot assays using phospho-specific antibodies against Akt to determine the level of Akt activation. A deletion mutant for Trib3 will be constructed to explore which domain of Trib3 may be responsible for causing death and modulating Akt phosphorylaiton and activation. Finally, my preliminary data suggest that Trib3 expression may be regulated by some of the transcriptional pathways that become activated during NGF withdrawal, namely the JNK, cell-cycle and FoxO pathways. This will be explored further by overexpression and knockdown of the transcription factors involved in these pathways and by measuring the level of Trib3 mRNA via quantitative PCR. PUBLIC HEALTH RELEVANCE: Disruption of NGF signaling not only results in developmental apoptosis, but also has been linked to loss of neurons observed in dementia, such as Alzheimer's disease. Therefore, NGF- withdrawal model is an excellent tool for exploring the factors leading to apoptosis. Trib3 is one such factor that requires thorough investigation and this type of research will make significant contribution toward identifying effective drug targets for treating neurodegenerative diseases.